Computational protocol: Cassava brown streak disease in Rwanda, the associated viruses and disease phenotypes

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Protocol publication

[…] The presence of cassava brown streak viruses was confirmed by sequencing a number of representative positive samples. Prior to sequencing, PCR‐amplified products were purified using a GeneJET PCR Purification kit (Thermo Scientific) following the manufacturer's instructions. Direct DNA sequencing in both directions was performed at the North Carolina State University Genomic Sciences Laboratory (Raleigh, USA). Sanger cycle sequencing reactions were performed using the BigDye termination mix (Applied Biosystems) and capillary sequencing on an ABI 3730xl DNA Analyzer (Applied Biosystems). Chromatography of sequences was viewed using the ApE program. Sequence quality control through sequence trimming and assembling was performed using CLC genomics workbench software. Quality scores of 0.05 were used for trimming, and sequences with scores rating below 50% were excluded from analyses. Alignment of sequences was performed using muscle in mega v. 7.Phylogenetic and molecular evolutionary relationships were examined by analysing partial coat protein‐encoding sequences (210 nt) of cassava brown streak viruses. For isolates of CBSV, the sequences started at nucleotide position 8653 to a stop codon with reference to isolate Kor 6 (acc. no. GU563327). For isolates of UCBSV, sequences started at nucleotide position 8841 to a stop codon with reference to isolate UG: Nam 23 (acc. no. FN434109). Analyses were conducted using mega v. 7. The Tamura & Nei substitution model (Tamura et al., ) was used for maximum likelihood (ML) tree reconstruction. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The analyses included 24 UCBSV and six CBSV Rwandan isolates characterized in this study (GenBank accession numbers KX168471 to KX168500) and published CBSV and UCBSV full genome sequences: one isolate of UCBSV (FJ039520) and one isolate of CBSV (NC012698) from Tanzania, three isolates of UCBSV (HG965222, FN434109 and NC014791) from Uganda, two isolates of UCBSV (KR911725, FN433930) and one isolate of CBSV (KR911737) from Kenya, one isolate of UCBSV (FN433933) from Malawi and one isolate of CBSV (FN434436) from Mozambique. The analysis also included Sweet potato mild mottle virus, genus Ipomovirus and family Potyviridae (NC003797) as an out‐group.To understand the genetic diversity of the isolates, estimates of evolutionary divergence over sequence pairs within and between groups were calculated. The analyses were conducted using the maximum composite likelihood model (Tamura et al., ). Also, the maximum likelihood estimate of transition/transversion bias (R) was calculated. The substitution pattern and rates were estimated under the Kimura (1980) 2‐parameter model (Tamura et al., ). Synonymous and nonsynonymous substitution rates influenced by fitness effect were estimated using snap v. 2.1. The analysis was performed on the web‐based platform (http://www.hiv.lanl.gov). […]

Pipeline specifications

Software tools CLC Genomics Workbench, MEGA-V
Applications Phylogenetics, GWAS
Organisms Manihot esculenta, Viruses, Ugandan cassava brown streak virus, Human poliovirus 1 Mahoney